No description available.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Dois estudos consecutivos foram realizados para reavaliar a faixa ideal de pH e a tolerÃncia de juvenis de tilÃpia do Nilo, Oreochromis niloticus, à acidez elevada da Ãgua de cultivo, em condiÃÃes eutrÃficas de cultivo. Os pesos iniciais dos animais eram semelhantes em ambas as fases. No primeiro trabalho, foram adotadas quatro condiÃÃes distintas de pH da Ãgua de cultivo: pH = 5,56  1,21 (pH 5); pH = 6,59  0,77 (pH 6); pH = 8,25  0,39 (pH 8); pH = 9,21  0,37 (pH 9), obtidas pelas aplicaÃÃes de soluÃÃes Ãcidas ou alcalinas à Ãgua. Os animais foram mantidos em tanques externos de 250 L por oito semanas. NÃo houve renovaÃÃo da Ãgua de cultivo ao longo de todo o trabalho. Neste trabalho, foram observadas variÃveis de qualidade de Ãgua, solo, zootÃcnicas e metabÃlicas. As maiores concentraÃÃes de nitrogÃnio amoniacal total da Ãgua foram encontradas nos tanques pH 5 e as menores nos tanques pH 8. A concentraÃÃo de gÃs sulfÃdrico na Ãgua foi maior nos tanques pH 5. O peso corporal final, a taxa de crescimento especÃfico e a produtividade do pescado nos tanques pH 5 foram maiores do que o observado nos tanques pH 9. Observou-se maior consumo de oxigÃnio nos respirÃmetros de pH 9. Concluiu-se que a faixa ideal de pH da Ãgua para o cultivo de juvenis de tilÃpia do Nilo, em Ãguas eutrofizadas, vai de 5 a 8. O delineamento experimental do segundo trabalho foi constituÃdo por quatro tratamentos, a saber: Ãguas de cultivo com diferentes valores de pH (pH 4: 4,12  0,84; pH 5: 5,13  0,74; pH 6: 6,14  0,64 e pH 8: 8,06  0,48), com cinco repetiÃÃes cada. NÃo houve troca da Ãgua de cultivo, apenas reposiÃÃo para manter o nÃvel inicial. Durante oito semanas, foram observadas variÃveis de qualidade de Ãgua, solo, zootÃcnicas, metabÃlicas e qualidade de efluentes. Embora a concentraÃÃo de nitrogÃnio amoniacal total (NAT) na Ãgua tenha sido menor nos tanques pH 8, sua concentraÃÃo de amÃnia nÃo-ionizada (NH3) foi maior que nos demais. Ao final, o menor peso corporal dos peixes foi observado nos tanques pH 8. Houve significativa melhora nos resultados de conversÃo alimentar e na taxa de eficiÃncia proteica com a acidificaÃÃo da Ãgua. As concentraÃÃes de NH3 nos efluentes dos tanques acidificados foram reduzidas. Concluiu-se que a acidificaÃÃo gradual da Ãgua de cultivo de juvenis da tilÃpia do Nilo atà pH 4 à benÃfica ao crescimento corporal dos peixes. A avaliaÃÃo de todos os resultados permite inferir que a faixa de pH da Ãgua para cultivo de O. niloticus seja de 4 a 8. / The first experiment aimed to reassess the Nile tilapiaâs optimum range of water pH for culture in eutrophic conditions. The initial weights of the animals were similar in both phases. There were four different conditions of water pH: pH = 5,56  1,21 (pH 5); pH = 6,59  0,77 (pH 6); pH = 8,25  0,39 (pH 8); pH = 9,21  0,37 (pH 9), that were obtained by acidic or alkaline applications upon the rearing waters. The fishes were maintained in twenty outdoor 250-L tanks for eight weeks. No water exchange was performed over the entire study. Variables of water and soil quality, growth performance and metabolism were monitored in the 1st work. The highest concentration of total ammonia nitrogen was observed in the pH 5 tanks and the lowest ones in the pH 8 ones. The hydrogen sulfide concentrations were higher in the pH 5 tanks. Fish final body weight, specific growth rate and yield were higher in the pH 5 tanks than in the pH 9 ones. There was a higher consumption of dissolved oxygen in the pH 9 respirometers than in the other ones. It was concluded that the optimum water pH range for Nile tilapia culture in eutrophic waters is 5 â 8. The second experiment aimed to determine the tolerance of Nile tilapia juveniles to high acidic culture waters and the effects of the culture water acidification upon the tanksâ effluents quality. The experimental design had four treatments, e.g., waters with different pH values (pH 4: 4,12  0,84; pH 5: 5,13  0,74; pH 6: 6,14  0,64 e pH 8: 8,06  0,48), with five replicates each. No water exchange was carried out over the entire study, just water replenishment to maintain the initial level. Variables of water quality, soil, growth performance, metabolism and effluents were observed over eight weeks. In spite of the lower total ammonia nitrogen (TAN) concentration in the pH 8 tanks, their levels of non-ionized ammonia (NH3) were the highest. At the end, the lowest fish body weight was observed in the pH 8 tanks. There was a significant improvement in the feeding conversion ratio and protein efficiency rate by the water acidification. The concentrations of NH3 were reduced in the acidic tank effluents. It was concluded that the gradual water acidification up to pH 4 is good to Nile tilapia juvenilesâ growth performance. The evaluation of all the results shows that the water pH range for cultivation of O. niloticus is 4-8.

Beim Bergbau werden bestehende Ökosysteme in großem Maße beeinflusst. Im ehemaligen Uranbergwerk Königstein (Sachsen) wurde die Umwelt durch den Einsatz von chemischen Säuren zur Lösung des Urans aus dem Erz (Laugung) in Folge der Verschiebung des pH-Wertes zusätzlich belastet. Durch diesen Prozess entstand eine Umgebung, die einen niedrigen pH-Wert und hohe Konzentrationen an gelösten Schwermetall-Ionen aufweist. Die komplexe mikrobielle Lebensgemeinschaft verschob sich daraufhin, indem sich bevorzugt säuretolerante und Schwermetall-tolerante Mikroorganismen durchsetzten. Diese Mikroorganismen wurden durch die Flutung der unter Tage Schächte im Jahr 2010 in ihrer Zusammensetzung erneut beeinflusst. In dieser Arbeit wurde die mikrobielle Biozönose nach Flutung der unter Tage Schächte des ehemaligen Uranbergwerkes Königstein charakterisiert und mit den Ergebnissen der mikrobiellen Diversität vor dem Flutungsprozess verglichen. Hierfür kam ein breites Spektrum an Methoden zum Einsatz, das klassische mikrobiologische Methoden und molekularbiologische Techniken umfasste. Die Analysen erfolgten dabei über mehrere Jahre hinweg, um die Variabilität der mikrobiellen Population im Grubenwasser planktonisch und im Biofilm zu erfassen.

Beim Bergbau werden bestehende Ökosysteme in großem Maße beeinflusst. Im ehemaligen Uranbergwerk Königstein (Sachsen) wurde die Umwelt durch den Einsatz von chemischen Säuren zur Lösung des Urans aus dem Erz (Laugung) in Folge der Verschiebung des pH-Wertes zusätzlich belastet. Durch diesen Prozess entstand eine Umgebung, die einen niedrigen pH-Wert und hohe Konzentrationen an gelösten Schwermetall-Ionen aufweist. Die komplexe mikrobielle Lebensgemeinschaft verschob sich daraufhin, indem sich bevorzugt säuretolerante und Schwermetall-tolerante Mikroorganismen durchsetzten. Diese Mikroorganismen wurden durch die Flutung der unter Tage Schächte im Jahr 2010 in ihrer Zusammensetzung erneut beeinflusst. In dieser Arbeit wurde die mikrobielle Biozönose nach Flutung der unter Tage Schächte des ehemaligen Uranbergwerkes Königstein charakterisiert und mit den Ergebnissen der mikrobiellen Diversität vor dem Flutungsprozess verglichen. Hierfür kam ein breites Spektrum an Methoden zum Einsatz, das klassische mikrobiologische Methoden und molekularbiologische Techniken umfasste. Die Analysen erfolgten dabei über mehrere Jahre hinweg, um die Variabilität der mikrobiellen Population im Grubenwasser planktonisch und im Biofilm zu erfassen.

No description available.

Acidophilic iron oxidizing bacteria of the betaproteobacterial genus “Ferrovum” are ubiquitously distributed in acid mine drainage (AMD) habitats worldwide. Since their isolation and maintenance in the laboratory has proved to be extremely difficult, members of this genus are not accessible to a “classical” microbiological characterization with exception of the designated type strain “Ferrovum myxofaciens” P3G. The present study reports the characterization of “Ferrovum” strains at genome and transcriptome level. “Ferrovum” sp. JA12, “Ferrovum” sp. PN-J185 and “F. myxofaciens” Z-31 represent the iron oxidizers of the mixed cultures JA12, PN-J185 and Z-31. The mixed cultures were derived from the mine water treatment plant Tzschelln close to the lignite mining site in Nochten (Lusatia, Germany). The mixed cultures also contain a heterotrophic strain of the genus Acidiphilium. The genome analysis of Acidiphilium sp. JA12-A1, the heterotrophic contamination of the mixed culture JA12, indicates an interspecies carbon and phosphate transfer between Acidiphilium and “Ferrovum” in the mixed culture, and possibly also in their natural habitat. The comparison of the inferred metabolic potentials of four “Ferrovum” strains and the analysis of their phylogenetic relationships suggest the existence of two subgroups within the genus “Ferrovum” (i.e. the operational taxonomic units OTU-1 and OUT-2) harboring characteristic metabolic profiles. OTU-1 includes the “F. myxofaciens” strains P3G and Z-31, which are predicted to be motile and diazotrophic, and to have a higher acid tolerance than OTU-2. The latter includes two closely related proposed species represented by the strains JA12 and PN-J185, which appear to lack the abilities of motility, chemotaxis and molecular nitrogen fixation. Instead, both OTU-2 strains harbor the potential to use urea as alternative nitrogen source to ammonium, and even nitrate in case of the JA12-like species. The analysis of the genome architectures of the four “Ferrovum” strains suggests that horizontal gene transfer and loss of metabolic genes, accompanied by genome reduction, have contributed to the evolution of the OTUs. A trial transcriptome study of “Ferrovum” sp. JA12 supports the ferrous iron oxidation model inferred from its genome sequence, and reveals the potential relevance of several hypothetical proteins in ferrous iron oxidation. Although the inferred models in “Ferrovum” spp. share common features with the acidophilic iron oxidizers of the Acidithiobacillia, it appears to be more similar to the neutrophilic iron oxidizers Mariprofundus ferrooxydans (“Zetaproteobacteria”) and Sideroxydans lithotrophicus (Betaproteobacteria). These findings suggest a common origin of ferrous iron oxidation in the Beta- and “Zetaproteobacteria”, while the acidophilic lifestyle of “Ferrovum” spp. may have been acquired later, allowing them to also colonize acid mine drainage habitats.

Acidophilic iron oxidizing bacteria of the betaproteobacterial genus “Ferrovum” are ubiquitously distributed in acid mine drainage (AMD) habitats worldwide. Since their isolation and maintenance in the laboratory has proved to be extremely difficult, members of this genus are not accessible to a “classical” microbiological characterization with exception of the designated type strain “Ferrovum myxofaciens” P3G. The present study reports the characterization of “Ferrovum” strains at genome and transcriptome level. “Ferrovum” sp. JA12, “Ferrovum” sp. PN-J185 and “F. myxofaciens” Z-31 represent the iron oxidizers of the mixed cultures JA12, PN-J185 and Z-31. The mixed cultures were derived from the mine water treatment plant Tzschelln close to the lignite mining site in Nochten (Lusatia, Germany). The mixed cultures also contain a heterotrophic strain of the genus Acidiphilium. The genome analysis of Acidiphilium sp. JA12-A1, the heterotrophic contamination of the mixed culture JA12, indicates an interspecies carbon and phosphate transfer between Acidiphilium and “Ferrovum” in the mixed culture, and possibly also in their natural habitat. The comparison of the inferred metabolic potentials of four “Ferrovum” strains and the analysis of their phylogenetic relationships suggest the existence of two subgroups within the genus “Ferrovum” (i.e. the operational taxonomic units OTU-1 and OUT-2) harboring characteristic metabolic profiles. OTU-1 includes the “F. myxofaciens” strains P3G and Z-31, which are predicted to be motile and diazotrophic, and to have a higher acid tolerance than OTU-2. The latter includes two closely related proposed species represented by the strains JA12 and PN-J185, which appear to lack the abilities of motility, chemotaxis and molecular nitrogen fixation. Instead, both OTU-2 strains harbor the potential to use urea as alternative nitrogen source to ammonium, and even nitrate in case of the JA12-like species. The analysis of the genome architectures of the four “Ferrovum” strains suggests that horizontal gene transfer and loss of metabolic genes, accompanied by genome reduction, have contributed to the evolution of the OTUs. A trial transcriptome study of “Ferrovum” sp. JA12 supports the ferrous iron oxidation model inferred from its genome sequence, and reveals the potential relevance of several hypothetical proteins in ferrous iron oxidation. Although the inferred models in “Ferrovum” spp. share common features with the acidophilic iron oxidizers of the Acidithiobacillia, it appears to be more similar to the neutrophilic iron oxidizers Mariprofundus ferrooxydans (“Zetaproteobacteria”) and Sideroxydans lithotrophicus (Betaproteobacteria). These findings suggest a common origin of ferrous iron oxidation in the Beta- and “Zetaproteobacteria”, while the acidophilic lifestyle of “Ferrovum” spp. may have been acquired later, allowing them to also colonize acid mine drainage habitats.:EIDESSTATTLICHE ERKLÄRUNG ... 2 CONTENT ... 4 SUMMARY ... 9 CHAPTER I ... 11 ORIGIN AND MICROBIOLOGY OF ACID MINE DRAINAGE ... 11 ACIDOPHILIC IRON OXIDIZING BACTERIA OF THE GENUS “FERROVUM” ... 12 APPLICATION OF OMICS-BASED APPROACHES TO CHARACTERIZE ACIDOPHILES ... 14 AIMS OF THE PRESENT WORK ... 15 CHAPTER II ... 17 ABSTRACT ... 18 INTRODUCTION ... 18 METHODS ... 19 GENOME PROJECT HISTORY ... 19 GROWTH CONDITIONS AND GENOMIC DNA PREPARATION ... 20 GENOME SEQUENCING AND ASSEMBLY ... 20 GENOME ANNOTATION ... 21 RESULTS ... 21 CLASSIFICATION AND FEATURES ... 21 GENOME PROPERTIES ... 24 INSIGHTS FROM THE GENOME SEQUENCE ... 24 COMPARATIVE GENOMICS ... 28 CONCLUSIONS ... 30 ACKNOWLEDGMENTS ... 32 AUTHOR CONTRIBUTIONS ... 32 CHAPTER III ... 33 ABSTRACT ... 34 INTRODUCTION ... 34 METHODS ... 36 ORIGIN AND CULTIVATION OF “FERROVUM” STRAIN JA12 ... 36 GENOME SEQUENCING, ASSEMBLY AND ANNOTATION ... 37 VISUALIZATION OF THE NEARLY COMPLETE GENOME ... 38 PHYLOGENETIC ANALYSIS ... 39 PREDICTION OF MOBILE GENETIC ELEMENTS ... 39 NUCLEOTIDE SEQUENCE ACCESSION NUMBER ... 39 RESULTS AND DISCUSSION ... 39 PHYLOGENETIC CLASSIFICATION OF “FERROVUM” STRAIN JA12 ... 39 GENOME PROPERTIES ... 40 NUTRIENT ASSIMILATION AND BIOMASS PRODUCTION ... 44 Carbon dioxide fixation ... 44 Central carbon metabolism ... 45 Nitrogen ... 47 Phosphate ... 49 Sulfate ... 50 ENERGY METABOLISM ... 50 Ferrous iron oxidation ... 50 Other redox reactions connected to the quinol pool ... 54 Predicted formate dehydrogenase ... 55 STRATEGIES TO ADAPT TO ACIDIC ENVIRONMENTS, HIGH METAL LOADS AND OXIDATIVE STRESS ... 55 Acidic environment ... 55 Strategies to cope with high metal and metalloid loads ... 58 Oxidative stress ... 59 HORIZONTAL GENE TRANSFER ... 60 CONCLUSIONS ... 61 ACKNOWLEDGMENTS ... 62 AUTHORS\' CONTRIBUTIONS ... 62 CHAPTER IV ... 63 ABSTRACT ... 64 INTRODUCTION ... 64 METHODS ... 66 ORIGIN AND CULTIVATION OF “FERROVUM” STRAINS PN-J185 AND Z-31 ... 66 GENOME SEQUENCING, ASSEMBLY AND ANNOTATION ... 66 PREDICTION OF MOBILE GENETIC ELEMENTS ... 67 COMPARATIVE GENOMICS ... 68 Phylogenomic analysis ... 68 Assignment of protein-coding genes to the COG classification ... 68 Identification of orthologous proteins ... 68 Comparison and analysis of genome architectures ... 69 RESULTS ... 69 GENERAL GENOME FEATURES AND PHYLOGENETIC RELATIONSHIP OF THE FOUR “FERROVUM” STRAINS ... 69 COMPARISON OF INFERRED METABOLIC TRAITS ... 71 Identification of core genes and flexible genes ... 71 Comparison of the central metabolism ... 74 Central carbon metabolism ... 74 Nitrogen metabolism ... 77 Energy metabolism ... 78 Cell mobility and chemotaxis ... 78 Diversity of predicted stress tolerance mechanisms ... 78 Maintaining the intracellular pH homeostasis ... 78 Coping with high metal loads ... 79 Oxidative stress management ... 79 IDENTIFICATION OF POTENTIAL DRIVING FORCES OF GENOME EVOLUTION ... 80 Prediction of mobile genetic elements ... 81 Linking the differences in the predicted metabolic profiles to the genome architectures ... 82 Gene cluster associated with flagella formation and chemotaxis in “F. myxofaciens” ... 84 Gene clusters associated with the utilization of alternative nitrogen sources ... 86 Gene cluster associated with carboxysome formation in “F. myxofaciens” and OTU-2 strain JA12 ... 87 Putative genomic islands in the OTU-strain JA12 ... 89 CRISPR/Cas in “F. myxofaciens” Z-31: a defense mechanism against foreign DNA ... 91 DISCUSSION ... 92 THE COMPARISON OF THEIR METABOLIC PROFILES INDICATES THE EXISTENCE OF OTU- AND STRAIN-SPECIFIC FEATURES ... 92 GENOME EVOLUTION OF THE “FERROVUM” STRAINS APPEARS TO BE DRIVEN BY HORIZONTAL GENE TRANSFER AND GENOME REDUCTION ... 94 Horizontal gene transfer ... 94 Mechanisms of genome reduction ... 95 CONCLUDING REMARKS ... 98 ACKNOWLEDGMENTS ... 98 AUTHOR CONTRIBUTIONS ... 98 CHAPTER V ... 99 ABSTRACT ... 100 INTRODUCTION ... 100 METHODS ... 102 CULTIVATION OF THE “FERROVUM”-CONTAINING MIXED CULTURE JA12 ... 102 Up-scaling of pre-cultures for the transcriptome study ... 103 Experimental setup of the transcriptome study ... 103 Cell harvest from large culture volumes ... 106 EXTRACTION OF TOTAL RNA ... 106 LIBRARY CONSTRUCTION AND SEQUENCING ... 107 DATA ANALYSIS ... 107 Processing of raw data ... 107 Quantification of gene expression levels ... 108 Functional analysis ... 108 RESULTS ... 108 CULTIVATION OF THE MIXED CULTURE JA12 IN THE MULTIPLE BIOREACTOR SYSTEM ... 108 Growth monitoring ... 108 Microbial composition ... 111 RNA SEQUENCING (RNA-SEQ) ... 112 FUNCTIONAL CATEGORIZATION OF EXPRESSED GENES ... 113 Functional assignment of highly expressed genes ... 117 Functional assignment of poorly expressed genes ... 121 COMPARISON OF EXPRESSION LEVELS OF GENES PREDICTED TO BE INVOLVED IN OXIDATIVE STRESS MANAGEMENT ... 122 DISCUSSION ... 124 METABOLIC PATHWAYS RELEVANT UNDER CULTURE CONDITIONS MIMICKING THE NATURAL CONDITIONS IN THE MINE WATER TREATMENT PLANT ... 125 Novel insights into the energy metabolism of “Ferrovum” sp. JA12 ... 125 Insights from poorly expressed genes ... 126 VARIATION OF GENE EXPRESSION PATTERNS UNDER THE DIFFERENT CONDITIONS ... 128 EVALUATION OF THE EXPERIMENTAL SET-UP INVOLVING THE MULTIPLE BIOREACTOR SYSTEM ... 129 CONCLUDING REMARKS: SIGNIFICANCE OF THE PRESENT TRANSCRIPTOME STUDY ... 130 ACKNOWLEDGMENTS ... 131 AUTHOR CONTRIBUTIONS ... 131 CHAPTER VI ... 133 ABSTRACT ... 133 EXTENDED INSIGHTS INTO THE FERROUS IRON OXIDATION IN BETAPROTEOBACTERIA ... 133 MECHANISMS OF PHYLOGENETIC AND METABOLIC DIVERSIFICATION WITHIN THE GENUS “FERROVUM” ... 136 INFERRED ROLES OF “FERROVUM” SPP. IN THE MICROBIAL NETWORK OF THE MINE WATER TREATMENT PLANT ... 138 PERSPECTIVES ... 143 REFERENCES ... 145 SUPPLEMENTARY MATERIAL ... 170 DATA DVD ... 170 SUPPLEMENTARY MATERIAL FOR CHAPTER III ... 171 NUCLEOTIDE ACCESSION NUMBERS ... 171 PHYLOGENETIC ANALYSIS ... 171 GENOME PROPERTIES ... 173 NUTRIENT ASSIMILATION ... 174 Carbon metabolism ... 174 FERROUS IRON OXIDATION ... 176 HORIZONTAL GENE TRANSFER ... 179 SUPPLEMENTARY MATERIAL FOR CHAPTER IV ... 180 PHYLOGENETIC ANALYSIS ... 180 ASSIGNMENT OF PROTEIN-CODING GENES TO THE COG CLASSIFICATION ... 180 COMPARISON OF THE CENTRAL METABOLISM ... 181 Predicted metabolic potential of the four “Ferrovum” strains ... 181 Genes predicted to be involved in the central metabolism, energy metabolism, cell motility and stress management in the four “Ferrovum” strains ... 183 PREDICTED MOBILE GENETIC ELEMENTS IN THE GENOMES OF THE FOUR “FERROVUM” STRAINS ... 184 THE FLAGELLA AND CHEMOTAXIS GENE CLUSTER ... 184 THE UREASE GENE CLUSTER ... 185 THE CARBOXYSOME GENE CLUSTER ... 186 PUTATIVE GENOMIC ISLANDS IN “FERROVUM” SP. JA12 ... 187 Gene content of the genomic islands ... 187 Flanking sites of the putative genomic islands 1 and 2 ... 188 SUPPLEMENTARY MATERIAL FOR CHAPTER V ... 189 ORGANIZATION AND OPERATION OF THE LABFORS 5 MULTIPLE BIOREACTOR SYSTEM ... 189 INVESTIGATION OF THE MICROBIAL COMPOSITION IN THE IRON OXIDIZING MIXED CULTURE JA12 ... 192 SUPPLEMENTARY DATA OF THE TRANSCRIPTOME DATA ANALYSIS ... 193 RNA-Seq statistics ... 193 Expression strength of protein-coding genes ... 194 Expression of genes involved in carboxysome formation ... 197 Expression of a ribosomal proteins-encoding gene cluster ... 199 Expression of a gene cluster presumably involved in ferrous iron oxidation ... 202 Lowest expressed genes ... 205 Expression of genes predicted to be involved in oxidative stress response ... 206 ACKNOWLEDGMENTS ... 208 COLLEAGUES ... 208 ERFOLGSTEAM “JUNGE FRAUEN AN DIE SPITZE” (“YOUNG WOMEN TO THE TOP“) ... 208 FAMILY AND FRIENDS ... 209 FUNDING ... 209 CURRICULUM VITAE ... 210 LIST OF PUBLICATIONS ... 212 RESEARCH ARTICLES ... 212 CONFERENCE PROCEEDINGS ... 212 ORAL PRESENTATIONS AND POSTERS ... 213